Despite advances in HIV-1 research and treatment, spread of the virus continues at a vigorous pace. A major contributing factor is cocaine, a popular recreational drug. Cocaine encourages high-risk behaviors that increase the chance of exposure to HIV-1. However, independent of behavioral factors, cocaine enhances the proliferation and transmission of the virus, and accelerates progression to AIDS. Our preliminary data suggest that cocaine and HIV-1 enhance the egress of HIV-1-enriched exosomes from host cells and enhance cell-tocell transmission of the virus. They appear to do this, at least in part, by modulating the expression and localization of tetherin, a key, anti-viral, host restrictio factor, and of LSP1, an actin-binding, cytoskeletal protein involved in modulating the innate immune response in DCs and tailoring the adaptive immune response to specific pathogens. The overall objective of this proposal is to elucidate the molecular mechanisms by which cocaine and HIV-1 partner to inhibit anti-HIV-1 host defenses and enhance the secretion and infectivity of HIV-1 exosomes. Specific points of innovation include: (1) focus on alterations in vesicular trafficking as a unique way HIV-1 and cocaine may subvert the action of host restriction factors; (2) characterize the novel involvement of LSP1 in the anti-viral activity of tetherin; (3) utilize cutting edge methodologies to isolate and characterize secreted exosomes; and (4) focus on alterations in the molecular composition and cargo of secreted exosomes as a novel mechanism by which HIV-1 and cocaine may enhance cell-to-cell transmission of HIV-1. The specific aims are: (1) characterize the tetherin/LSP1 complex, the role of LSP1 in tetherin-mediated anti-HIV-1 activities, and how HIV-1 Vpu reduces LSP1 expression, (2) characterize the effects of cocaine on tetherin expression, the tetherin/LSP1 complex, HIV-1 trafficking, and NF-B signaling, and (3) characterize the effects of HIV-1 and cocaine on the differential composition and cargo of exosomes released from DCs and T-cells, and the effects of these exosomes on anti-viral host restriction factors. By generating these new data, we hope to provide the foundation for innovative strategies to prevent or treat HIV-1 infection more effectively in the growing population of cocaine users.